An unmet need for expansion of primary cells and progenitor cells in three dimensions (3-D) is an extracellular matrix (ECM) substitute with user-controllable composition and compliance to which ECM proteins or synthetic peptides can be covalently attached by the end user. Glycosan BioSystems currently markets HyStem, an in situ-crosslinkable, semi-synthetic ECM composed of poly(ethylene glycol) diacrylate (PEGDA) and crosslinked thiol-modified hyaluronan (Glycosil) for culture of a variety of pluripotent cell types. The elastic modulus of the matrix determines to a large extent cell fate, and can be varied by changing crosslink density and concentration. In Phase I we will test the feasibility of a simple system for attachment of proteins to the hydrogels that would permit any synthetic polypeptide or any native matricellular protein (MP) to be covalently attached to the thiol-modified HA component of HyStem. In contrast to adding native ECM proteins that are not bound to the gel, and thus cannot provide mechanical support, this approach directly couples the "pull" of the cells on the proteins to the elastic modulus of the gel. We will compare MP-attached gels with the gelatin-containing HyStem-C hydrogel to identify conditions that maintain quiescence of mesenchymal stem cell (MSC) and that improve MSC differentiation capacity into adipocytes. Following the optimization of protein attachment conditions, we will determine the feasibility of this approach for growing MSCs in or on HyStem- MP hydrogels as autologous, animal-free feeder layers for the culturing of CD34+ stem cells derived from bone marrow. PUBLIC HEALTH RELEVANCE: An unmet need for expansion of primary cells and progenitor cells in three dimensions (3-D) is an extracellular matrix (ECM) substitute with user-controllable composition and compliance to which ECM proteins or synthetic peptides can be covalently attached by the end user. Glycosan BioSystems currently markets HyStem", an in situ-crosslinkable, semi-synthetic ECM composed of poly(ethylene glycol) diacrylate (PEGDA) and crosslinked thiol-modified hyaluronan (Glycosil") for culture of a variety of pluripotent cell types. The goal of this proposal is to test the feasibility of a simple system for covalent attachment of extracellular matrix proteins to the HyStem hydrogel and to tailor the protein composition and stiffness for modulating the quiescence or differentiation of human mesenchymal stem cells.